Recombinant DNA technology has permitted the expression of heterologous protein in host cells such as E. coli bacteria. In the case of the growth hormone somatotropin, the protein is sequestered in refractile bodies within the cytoplasm of the host cells. The refractile bodies may be recovered from the host cell culture by disrupting the cell so as to release the refractile bodies, and thereupon collecting the refractile bodies as a solid pellet by differential centrifugation. The refractile bodies are solubilized in an aqueous solution of a suitable chaotropic agent such as urea or guanidine hydrochloride at an alkaline pH, generally in the range of 9-12. The solubilized proteins are subsequently naturized by contact with a mild oxidizing agent to form intramolecular disulfide bonds while refolding the protein to its biologically active native conformation. Methods for the solubilization and the naturation of somatotropin protein produced by E. coli bacteria using recombinant DNA technology are described in U.S. Pat. No. 4,511,502 and U.S. Pat. No. 4,652,630, each of which is incorporated herein by reference.
The monomeric form of somatotropin is the most biologically active, and the major yield loss occurring during the solubilization and naturation process is due to the formation of dimer and higher aggregate forms of the protein which are largely removed during further processing and purification of the monomer. Reducing the initial formation of these impurities would not only improve initial monomer yields but also reduce subsequent product losses during the monomer recovery and purification steps.
It is accordingly an object of the present invention to provide an improved process for the solubilization and naturation of somatotropin. It is a further object of the invention to provide an improved process resulting in higher yields of somatotropin monomer during the solubilization and naturation phase of the process. It is a further object of this invention to provide an improved process to reduce the formation of dimer and aggregate forms of somatotropin during the solubilization and naturation thereof. It is a still further object of this invention to improve yields of somatotropin monomer recovered from refractile bodies produced by recombinant DNA technology. These and other objects of the present invention will be apparent from the ensuing description and claims.